Development of Anchor Loci to Map a BT Resistance Factor in the Domesticated Silkworm, Bombyx mori

The BT toxin is an insecticide produced by the bacterium Bacillus thuringiensis, and millions of acres of genetically modified crops expressing this agent have been planted. However, with such widespread use, there is a great potential for pests resistant to this toxin to develop. An understanding of how BT resistance operates and is inherited is central to ensuring continued use of the transgenic crops. Bombyx mori, the domesticated silkworm, is the model organism for the Lepidoptera, an order of insects containing many important agricultural pests. Screening shows that BT resistance is present in some silkworm strains, and linkage group 15 (LG15) harbors a major BT resistance factor (W. Hara, personal communication). We have found BT resistance in a different silkworm strain, and are testing to determine if the same linkage group is responsible. To do this we chose ribosomal protein (Rp) genes, which also mapped to this chromosome, as candidates for genetic markers. Because these genes are orthologous, meaning that they are present in related species and are derived from the same ancestor, they are considered to be anchor loci, which can be applied to a variety of species. By comparing genomic and cDNA sequences of Rp genes, I identified introns, likely to be the most polymorphic areas of a gene, for amplification by polymerase chain reaction. Using a mapping panel prepared by a graduate of the Goldsmith lab, analysis with one anchor locus suggests that the resistance phenotype does indeed map to LG15. More markers that have been mapped to this chromosome are being scanned in an effort to corroborate this finding. Recent publications have shown that there is strong synteny, the conservation of chromosomal linkage of orthologous genes, within the Lepidoptera. As such, it is expected that these anchor loci will be present on the same linkage group as the putative BT resistance factor in related species, and therefore of use to future studies

Repression of Mitochondrial Translation, Respiration and a Metabolic Cycle-Regulated Gene, SLF1, by the Yeast Pumilio-Family Protein Puf3p

Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3′-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing

Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p

The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay. Using integrated genome-wide approaches we define an expanded set of Puf3p target mRNAs and quantitatively assessed the global impact of loss of PUF3 on gene expression using mRNA and polysome profiling and quantitative proteomics. In agreement with prior studies, our sequencing of affinity-purified Puf3-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. Additionally, we also found 720  new mRNA targets that predominantly encode proteins that enter the nucleus. Comparing transcript levels in wild-type and puf3∆ cells revealed that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only a limited subset of its target mRNAs. Finally, proteomic and translatomic studies suggest that loss of Puf3p has widespread, but modest, impact on mRNA translation. Taken together our integrated multi-omics data point to multiple classes of Puf3p targets, which display coherent post-transcriptional regulatory properties and suggest Puf3p plays a broad, but nuanced, role in the fine-tuning of gene expression

Analysis of <i>puf3Δ</i> Yeast Strains.

<p>Analysis of wild-type (wt) and an isogenic <i>puf3Δ</i> strains are shown. (A) Mitochondrial oxygen consumption assayed at 12 hours (log phase), 24 hours (early stationary phase), and 48 hours post-inoculation (1 day into stationary phase). The bars represent the mean ± SD with p-values as indicated (*<0.05, **<0.01, ***<0.001). Statistical analysis was performed with Prism 5 software, using a Student's t-test (unpaired, two-tailed). (B) Western blot of indicated proteins in log-phase growth (representative of three biological replicates). (C) Autoradiogram of separated mtDNA-encoded proteins labeled with ³⁵S-methionine and ³⁵S -cysteine at logarithmic growth. A section of the same gel stained with coomassie blue to demonstrate loading is shown underneath. (D) mtDNA copy number.</p

Comparison of mitochondrial parameters in <i>puf3Δ</i>, <i>tor1Δ,</i> and <i>puf3Δ tor1Δ</i> mutant strains.

<p>(A) Mitochondrial oxygen consumption during early log phase. (B) Western blot analysis of protein extracts derived from log phase cultures (representative of three biological replicates). (C) Steady-state levels of <i>COX17</i>, <i>COX12</i>, and <i>RIP1</i> transcripts in log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020441#pone-0020441-g001" target="_blank">Figure 1</a>.</p

The Regulation of Respiration by Puf3p is Independent of Mitochondrial Biogenesis Pathways.

<p>Shown is the analysis of wild-type (wt) and <i>puf3Δ</i> strains with plasmids over-expressing <i>HAP4</i> and <i>GSM1</i> (<i>HAP4 GSM1</i>) and corresponding empty vectors (vectors) (A) Mitochondrial oxygen consumption during late-log phase of growth. (B) Western blot analysis (representative of three biological replicates). (C) Steady-state levels of <i>COX17</i>, <i>COX12</i>, and <i>RIP1</i> transcripts in late log phase of growth. The values indicate the mean +/− SD with p-values indicated as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020441#pone-0020441-g001" target="_blank">Figure 1</a>.</p

Comparison of mitochondrial parameters in wild-type and <i>puf3Δ</i> mutant strains treated with the TORC1 inhibitor rapamycin.

<p>(A) Mitochondrial oxygen consumption of cells treated with rapamycin during early log phase and analyzed during late log phase. (B) Western blot analysis of protein extracts derived from late log phase cultures (representative of three biological replicates). (C) Steady-state levels of <i>COX17</i>, <i>COX12</i>, and <i>RIP1</i> transcripts in late log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020441#pone-0020441-g001" target="_blank">Figure 1</a>.</p

Over-expression of Slf1p increases respiration and CLS, and expression of Sls1p is repressed by Puf3p.

<p>(A) <i>SLF1</i> DNA sequence depicting the stop codon (bold) and Puf3 element (underlined). (B) Mitochondrial oxygen consumption of wild-type cells carrying an empty vector (YEp351) or YEp351 with <i>SLF1</i> during early stationary phase. (C) Western blot analysis of protein extracts derived from the same strains in (B) (representative of three biological replicates). (D) CLS analysis of the same strains in (B). (E) Q-RT-PCR analysis <i>COX17</i>, <i>COX12</i>, and <i>RIP1</i> transcript levels at early stationary phase from the same strains in (B). Analysis of Slf1p levels by western blot (F) and <i>SLF1</i> mRNA levels by RT-PCR (G) in wild-type and <i>puf3Δ</i> strains in log-phase growth (representative of three biological replicates).</p